Microtiter plate monoclonal antibody epitope analysis of Ca2+- and Mg2+-induced conformational changes in troponin C

Abstract

Spectroscopic methods such as circular dichroism and Förster resonance energy transfer are current approaches to monitoring protein conformational changes. Those analyses require special equipment and expertise. The need for fluorescence labeling of the protein may interfere with the native structure. We have developed a microtiter plate-based monoclonal antibody (mAb) epitope analysis to detect protein conformational changes in a high throughput manner. This method is based on the concept that the affinity of the antigen-binding site of an antibody for the specific antigenic epitope will change when the 3-D structure of the epitope changes. The effectiveness of this approach was demonstrated in the present study on troponin C (TnC), an allosteric protein in the Ca 2+ regulatory system of striated muscle. Using TnC purified by a highly effective rapid procedure and mAbs developed against epitopes in the N-and C-domains of TnC Enzyme-linked immunosorbant assay (ELISA) clearly detected Ca 2+ -induced conformational changes in both the N-terminal regulatory domain and the C-terminal structural domain of TnC. On the other hand, Mg 2+ -binding to the Cdomain of TnC resulted in a long-range effect on the N-domain conformation, indicating a functional significance of Ca 2+ -Mg 2+ exchange at the C-domain metal ion-binding sites. In addition to further understanding of the structure-function relationship of TnC, the data demonstrate that the mAb epitope analysis provides a simple high throughput method for monitoring 3-D structural changes in native proteins under physiological condition and has broad applications in protein structure-function relationship studies. Keywordsepitope affinity analysis; monoclonal antibody; ELISA; protein conformation; troponin C; Ca 2+ -regulationThe changes in protein three-dimensional (3-D) 1 structure are commonly examined by spectroscopic methods such as circular dichroism and Förster resonance energy transfer (FRET). These methods need to be performed in expert laboratories and require highly specialized equipment. Spectroscopic methods often require external labeling of the protein, which may interfere with the native structure. Therefore, a more readily accessible method would be desirable for detecting changes in protein 3-D structure.

To Investigate Protein Evolution by Detecting Suppressed Epitope Structures

“…ELISA epitope analysis (Wang and Jin, 1998; Jin et al, 2007) was employed to compare the relative binding affinity of the antibody epitope probes to the isoforms and fragments of TnI and TnT for their relationships in three dimensional structure. The purified TnI or TnT protein was dissolved in Buffer A (0.1 M KCl, 3 mM MgCl 2 , 10 mM PIPES, pH 7.0) at 2 μg/mL and used at 100 μL/well to non-covalently coat 96-well microtiter plates by incubation at 4 °C overnight.…”

“…In addition to detecting a static structure, antibodies against allosteric epitopes provide probes for measuring conformational changes in a protein (Wang and Jin, 1998; Jin et al, 2000a; 2000b). Protein epitopic structures can be quantitatively studied by microtiter plate enzyme-linked immunosorbant assay (ELISA) that effectively detects conformational differences and changes under native conditions (Wang and Jin, 1998; Jin et al, 2007). …”

The heart-specific NH2-terminal extension regulates the molecular conformation and function of cardiac troponin I

“…mAbs provide reagents for recognizing specific epitopes and can be used in monitoring protein conformation, structural dynamics, and folding (18). We have applied antibody probes against epitopes in allosteric proteins to detect conformational differences and changes using high throughput microtiter plate ELISA under native conditions (23,24,27,39).…”

“…The wells were then washed with buffer T at pCa 4 or pCa 9. The binding of TnC to immobilized TnI was detected using an anti-TnC mAb 2C3 (24), and the ELISA steps were the same as above, except that the washing and antibody dilution buffers were maintained at either pCa 4 or pCa 9.…”

“…Bovine cardiac TnT was purified from adult ventricular muscle as described previously (25). Chicken fast TnC was expressed from cloned cDNA and purified as described previously (24). Microtiter plates were coated as above except that the cTnI proteins were at a concentration of 2.5 g/ml and the coating buffer A contained 1 mM DTT.…”